Background
In 2014, 9.6
million people became infected with tuberculosis (TB) disease, and 1.5 million
died from the disease.2 Figure 1 shows the number of new TB cases in
2013. New cases of TB emerge in almost all countries in the world every year,
especially the global south, where the TB incidence rates can be as high as
more than 500 per 100,000 population.
Figure 1. Estimated
TB incidence rates in 20133
The TB epidemic is very daunting,
partly because the bacterium that causes the disease, Mycobacterium tuberculosis, is extremely virulent. However,
scientists believe that, just like the Greek hero Achilles (whose vulnerable
heel is his fatal weakness), M.
tuberculosis also has its deadly weakness.
Figure 2. Heels are
Achilles’ kryptonite4
M.
tuberculosis’s virulence mostly comes from its extremely thick lipid cell
wall, which makes the cell difficult to kill.5 The bacterium is
known to use host cell cholesterol as its main carbon source; however, the
breakdown of cholesterol can lead to the generation of propionyl-CoA, which can
produce potentially toxic intermediates.
M.
tuberculosis has three different pathways to deal with propionyl-CoA
toxicity. The first pathway is called the methylcitrate cycle (MCC), which
utilizes a protein called ICL1 that allows for the conversion of propionyl-CoA
to succinate and pyruvate, which can then be used in the TCA cycle. The second
pathway is called the methylmalonyl pathway (MMP), which requires vitamin B12;
methylmalonyl-CoA (MM-CoA) enters the MMP. MM-CoA can be produced by
propionyl-CoA carboxylase. The third pathway, and the one focused on in this
paper, is the pathway that incorporates propionyl-CoA intermediates into the
cell’s lipid cell wall.
Propionyl-CoA
can be used to build the cell wall by being turned into MM-CoA, which are then
used as building blocks of methyl-branched long-chain fatty acids like
phthiocerol dimycocerosates (PDIM) and sulfolipid-1 (SL-1).
The three pathways
In this study, Lee et al. showed that M. tuberculosis uses host-cell lipid stores to build up its thick
lipid cell wall, thereby reducing propionyl-CoA toxicity while contributing to
its virulence. In their experiments, Lee et
al. artificially induced propionyl-CoA synthesis by adding propionate to
their culture medium; propionate is converted into propionyl-CoA by the enzyme
encoded for by gene acs(Rv3667).
Thus, “propionate toxicity” is often used interchangeably with “propionyl-CoA
toxicity.” Many of their initial experiments involved a mutant of M. tuberculosis that did not have the
ICL1 gene, which we will refer to as the ∆icl1 mutant. Recall
that the ICL1 protein is necessary for the MCC to run. That means these mutants
could not utilize the MCC to rid itself of propionyl-CoA toxicity.
In one of their initial experiments, researchers cultured the
∆icl1 mutant in the presence of propionate and absence of vitamin B12, and
measured the amount of bacterial growth through optical density. Because the
mutant was unable to rid itself of propionyl-CoA toxicity via the MCC and MMP,
the mutant grew poorly in the presence of propionate; however, their growth
could be rescued by adding in a supplement, acetate, as shown in Figure 3.
Figure 3. Fatty acids
(acetate in this example) are needed for MB lipid synthesis
Fatty acid rescue
Researchers next wanted to
determine if longer-chain fatty acids (recall that acetate is extremely short;
only two carbons) could rescue growth in the ∆icl1
mutants. While it was known that intermediate length fatty acids were toxic to
the cell (those containing 10-16 carbons) from a previous study,4
shorter chain fatty acids (C2-C8) and long-chain fatty acids (C18-C24) were
able to rescue the ∆icl1 mutants from
propionate toxicity. This led researchers to believe that since the ∆icl1
mutants could not rid the cell of propionyl-CoA via the MCC or MMP (recall that
there is no vitamin B12 supplemented here), the fatty acids must be playing a
role in the incorporation of methylmalonyl-CoA, a product of propionyl-CoA,
into the PDIM to deal with propionyl-CoA toxicity. This was supported by
previous work which found that there needs to be 4:1 ratio of methylmalonyl-CoA
to acyl primer, which can be produced from the fatty acids, to synthesize PDIM
in mycoserosic acid.
To test
this hypothesis, they used both the ∆icl1 mutants and the
Erdman strain, a high PDIM-producing but otherwise wild-type strain of M. tuberculosis. They then labeled the
two strains with 3H and 14C isotopes in propionate,
acetate, and a long-chain fatty acid known as stearic acid. The isotopes are
heavier and can therefore be identified by thin layer chromatography (TLC).
After growing the strains in the presence of propionate and acetate, the
researchers concluded that the acetate was in fact incorporated into the PDIM
of both cell types. Stearic acid, a long-chain fatty acid, was also seen in the
PDIM of both cell types in the presence of propionate, indicating that the
entire chain was incorporated into the PDIM to act as an acyl primer. This was
seen in the gel in figure 4, with the red boxes emphasizing bands created by
the ∆icl1 mutants and blue boxes emphasizing bands created
by the Erdman strain; regardless of whether propionate, acetate, and/or stearic
acid were tagged, the tag was seen in the PDIM bands, indicating it had been
incorporated into the PDIM.
Figure 4. TLC
analysis of cell lipids, showing incorporation of propionate, acetate, and
stearic acid into PDIM
Lipid droplet rescue
Interestingly, researchers also
found that M. tuberculosis ∆icl1
mutants and mutants unable to metabolize cholesterol survived in the macrophage
at a lower rate than the wild-type bacterium. They hypothesized that the
decreased survival rate of both mutants may have resulted from a similar
defect; namely, researchers predicted that the inability of these mutants to
use lipid droplet stores in its host cell was decreasing their fitness. To test
their hypothesis, researchers induced lipid droplet formation in host
macrophages by adding oleate, a long-chain fatty acid, into its culture medium.
The lipid droplet-induced macrophages and a control were infected with the ∆icl1
mutant. The growth of the ∆icl1 mutant was
measured using fluorescence and cfu (colony forming unit; basically, they lysed
all the macrophages and plated a sample from each macrophage, then counted how
many colonies grew on the resulting plates) counts (Figure 5).
The M. tuberculosis ∆icl1 mutants that
infected macrophages with greater lipid stores grew significantly better than
the mutants grown in untreated macrophage. Lee et al. compared these results to the rescue of the ∆icl1
mutants treated with vitamin B12 (which helps remove propionyl-CoA through the
MMP) as shown in Figure 5. They found that when either pathway was available,
the mutants were able to grow much better than when neither pathway was
available.
Figure 5. In a Δicl1 mutant lacking vitamin B12,
addition of fatty acids (oleate in this case) rescues the TB bacteria [LM1] .
They next wanted to determine if
the fatty acids from the host lipid stores were rescuing the M. tuberculosis ∆icl1
mutants by allowing them to use the host fatty acids as the acyl primer
necessary for methylmalonyl-CoA to be incorporated into their PDIM. To test
this, they induced host lipid stores in the macrophages with radioactive
isotopes of different fatty acids. If the isotopes were incorporated into the
PDIM of the cell, this would indicate that M.
tuberculosis was in fact accessing the host’s fatty acid stores that could
act as an acyl primer in the PDIM. They then extracted the PDIM from the M. tuberculosis ∆icl1
mutants and found traces of the carbon isotopes, providing evidence for their
hypothesis.
The genes responsible
Lee et al. attempted to validate and better understand their results by
using a forward genetic screen, which means they attempted to find the genes
that would produce the phenotypes they found. They used a TraSH (transposon
site hybridization) screen, in which the researchers mutate one gene per
bacterium, and then allow them to grow. Then, they see which bacteria died and
which ones lived under certain conditions, and figure out which genes were
mutated in those bacteria.3
Lee et al. conducted a series of tests that would help them learn about
the genes involved in each of the three pathways. For example, they looked at M. tuberculosis mutants that had
mutations in genes involved in the synthesis of non-methyl-branched lipids (lysX, plsC, mbtB, and mbtK are all genes that encode for
enzymes involved in this), and they saw that these mutants actually had better
growth in the presence of propionate. The researchers reasoned that because the
synthesis of non-methyl-branched lipids requires long-chain fatty acids and
these mutants could not create these lipids, they could use all of the
available long-chain fatty acids to create fatty acid primers for biosynthesis
of methyl-branched lipids to incorporate into the cell wall and reduce
propionyl-CoA toxicity more than the wild-type M. tuberculosis. Other tests like this one allowed the researchers
to create a map of all of the genes involved in the pathway that incorporates
propionyl-CoA into PDIM (Figure 6).
Figure 6. Map of
genes involved in assimilation of propionate and fatty acids into
methyl-branched lipids
Conclusions and
future studies
In this study, Lee et al. developed a model that elucidates
the interplay among the three major pathways of propionyl-CoA metabolization.
More specifically, their experiments focused on the last pathway and determined
how M. tuberculosis utilizes its
hosts lipid stores to help remove propionyl-CoA toxicity while contributing to
its thick cell wall (Figure 7).
Figure 7. Proposed
model of assimilation of host lipid stores into M. tuberculosis cell wall
As shown in Figure 7, propionyl-CoA
and acetyl-CoA are generated from cholesterol catabolism. If the MCC is intact,
propionyl-CoA can be catabolized into succinate and pyruvate, feeding into the
TCA cycle. However, if the MCC is inhibited, propionyl-CoA is catabolized to
toxic intermediates. One compensatory pathway to decrease the propionyl-CoA
toxicity is the MMP, which generates methylmalonyl-CoA from propionyl-CoA,
which can be used in the TCA cycle in the presence of vitamin B12. When the MCC
is halted and vitamin B12 is unavailable, propionyl-CoA and acyl primers
generated from fatty acids are incorporated into methyl-branched cell wall
lipids.
The researchers’ experimental
results confirmed that both acetate and vitamin B12 rescue the Δicl1 mutant, consistent with the
proposed three-pathway model of propionate metabolism. They also found that
while short-chain fatty acids undergo beta-oxidization to yield acetyl-CoA and
further become acyl primers to synthesize cell wall lipids, long-chain fatty
acids can bypass the beta-oxidation step and the entire fatty acid can be
incorporated into the cell wall. In vitro
studies found that M. tuberculosis
can utilize lipid droplets in the host cells to alleviate propionate-related
stress.
For the
future, it would be interesting to do experiments that would allow us to use
this new information to help reduce virulence of M. tuberculosis in humans. We thought about cutting out specific
parts of the pathway where propionyl-CoA,, fatty acids, and cholesterol are incorporated
into the cell wall. For example, what if we reduced the amount of cholesterol
in the host cell? Would the M.
tuberculosis not be as virulent because there is less cholesterol available
to help fortify its cell wall? We also think it would be interesting to see if
feeding a host cell intermediate fatty acids is toxic to M. tuberculosis, since we know in general that the intermediate
fatty acids cannot be incorporated into the cell wall.
This study
uncovered a potential M. tuberculosis
“Achilles heel”; as the delicate balance of the three pathways helps the cell
when it is attempting to get nutrients from the lipid stores in the host cell.
It is possible that by messing up said delicate balance can cause the cell to
be less virulent, since one of the biggest reasons M. tuberculosis is so virulent is due to its thick cell wall. The
results from this study could potentially be the beginning of an effective way
to rid people of tuberculosis disease by diminishing M. tuberculosis virulence.
References
1.
Lee, W., Vanderven, B., Fahey, R., & Russell, D.
(2013). Intracellular Mycobacterium tuberculosis Exploits Host-derived Fatty
Acids to Limit Metabolic Stress. Journal
of Biological Chemistry, 288(10),
6788-6800. Retrieved November 22, 2015, from US National Library of Medicine
National Institutes of Health.
2.
Tuberculosis. (n.d.). Retrieved November 24, 2015,
from
http://www.who.int/mediacentre/factsheets/fs104/en/
3.
Global Tuberculosis Report 2014, World Health
Organization
4.
http://charactertherapist.blogspot.com/2013/08/how-to-develop-your-characters-achilles.html
5.
Winstead, E. (n.d.). Watching Genomes Work. Retrieved
November 24, 2015, from http://www.genomenewsnetwork.org/articles/11_01/Watching_genomes.shtml
6.
Dubos, R. J. "The Effect Of Organic Acids On
Mammalian Tubercle Bacilli." Journal of Experimental Medicine 92.4 (1950):
319-32. Web.
7.
Massoni, Shawn. (2015). Bacterial Cell Wall and Other
Layers. Mount Holyoke College.
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